PROJECT SUMMARY Carbohydrate recognition is one of the most sophisticated recognition processes in biological systems. The exceptional capacity of oligosaccharides for high density information coding is essential for cells to mediate many important aspects of cell-cell recognition. The endogenous glycan-binding proteins, known as lectins, have been increasingly recognized as ?decoders? of the carbohydrate-encoded biological information based on their ability to distinguish between closely related glycan structures. This inherent complexity constitutes a major barrier to understanding the functional significance of these modifications on the molecular level. To help address this knowledge gap, the proposed research aims to characterize the interaction between Tn/sTn glycans of tumor-associated MUC1 with macrophage galactose-specific lectin (MGL). To date, the significance of the diversity in the type and positions of tumor-associated O-glycans on modulation of immune responses by MGL is still unclear. Thus, mono- and multiple-glycosylated MUC1 peptide models and MUC1-derived glycopeptide positional scanning combinatorial libraries displaying native-like heterogeneous and aberrant Tn/sTn O-glycan epitopes will be prepared to examine epitope heterogeneity, the glycoside cluster effect, and steric hindrance effect of neighboring glycans on binding to lectins (Aim 1). Individual glycopeptides with enhanced ability to bind MGL will be synthesized in a multivalent ligand display to mimic biological polyvalent systems (Aim 2). The effect of the central scaffold (linear or branched), presenting multiple copies of the recognition elements, on the lectin binding specificities will be evaluated by isothermal titration calorimetry (ITC) and atomic force spectroscopy (AFM) (Aim 3). The thermodynamics of ligand-receptor binding interaction will be determined by ITC. Since the mechanical properties of the receptor-ligand complexes cannot be obtained solely from thermodynamic measurements, but rather from the reaction path that leads to complex separation, single-molecule AFM will be used to determine the binding kinetics of the interaction. An important component of this proposal is to engage students actively in interdisciplinary research. We will offer a hands-on research experience for undergraduate and graduate students on the interface of chemistry and biology, giving students a unique experience that brings together many aspects of synthetic organic chemistry, glycochemistry and glycobiology as emphasized in the recent report from the National Research Council of the U.S. National Academies. Our goal is to broaden the future community of researchers with an emphasis on increasing the participation of women and minorities in chemistry and other STEM disciplines. In summary, this project will provide novel and unique synthetic tools to study the effects of glycans on protein structure and to decipher the biochemical basis of glycan?protein interactions in regulation of the immune system.